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作者(中文):鄒安洋
作者(外文):ZOU, AN-YANG
論文名稱(中文):利用CRISPR/Cas13抑制lncRNAs表現促使間葉幹細胞往硬骨分化
論文名稱(外文):CRISPR/Cas13 for suppressing lncRNAs to Promote Osteogenesis Differentiation of Mesenchymal Stem cells
指導教授(中文):胡育誠
指導教授(外文):HU, YU-CHEN
口試委員(中文):黃振煌
林進裕
口試委員(外文):Huang, Jen-Huang
Lin, Chin-Yu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學號:107032544
出版年(民國):109
畢業學年度:108
語文別:中文
論文頁數:64
中文關鍵詞:CRISPR間葉幹細胞骨分化長非編碼RNA
外文關鍵詞:CRISPRMesenchymal Stem cellOsteogenesislong non-coding RNA
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大範圍的頭蓋骨缺陷在目前臨床骨科醫學上是一個難以克服的難題。而long non-coding RNAs (lncRNAs)在幹細胞分化過程中扮演重要角色,過去研究上常以siRNA系統調控RNA表現,但其專一性較差常伴隨著off-target效應產生,且其調控效率仍有提升空間,近年發現的CRISPR/Cas13系統為原生RNA-guided RNA-binding核酸酶系統,可有效調控RNA,且僅需建構小片段crRNA即可調控多種基因,因此本篇使用此系統調控參與分化之lncRNAs表現。
文獻指出lncRNA SRA與PU.1 AS在細胞向脂肪分化時表現量會上升。另外,當lncRNA MIAT表現受到抑制,ASCs將會往硬骨細胞分化。因此,同時抑制多種lncRNAs,預期可以抑制脂肪/骨髓間葉幹細胞往脂肪分化同時促進細胞往硬骨分化。
實驗結果發現在rASCs中僅有Cas13d蛋白表現即可抑制lncRNAs的表現,而兩週後定性實驗中亦觀察到僅有Cas13d蛋白組別與Cas13d/gRNA的組別相較於Mock組,細胞中油滴累積較少與更為明顯的礦化程度,顯示被調控的lncRNAs是可能源自Cas13d而非Cas13d/gRNA。而經過測試不同細胞株,同時將control改以Cas13d/non-targeting gRNA,最後選擇mBMSCs作為後續實驗細胞株。由結果發現,CRISPR/Cas13d可以有效抑制lncRNAs與脂肪分化指標基因PPARγ與CEBPα,同時亦可提升硬骨分化指標基因Runx2、OSX、ALP、OPN的表現。但在Oil Red O染色中,NT仍可有效減少油滴累積,顯示其可能仍潛藏off-target效應。
Today critical-sized calvarial bone-defect repair remains a challenging task. Long non-coding RNAs (lncRNAs) play an important role in stem cells differentiation. In the past, they usually use siRNA system to regulate RNA expression, but its specificity isn’t enough and cause some off-target effect. Recently, CRISPR/Cas13 system is introduced, it is natral RNA-guided RNA-binding nucleic acid system. It can effectively regulate RNA, and only contruct some small crRNA to regulate multiple genes. So we develop this system to regulate lncRNAs involved in differentiation.
The expression of lncRNA SRA and PU.1 AS will increase during adipogenesis. In addition, lncRNA MIAT is downregulated during osteogenesis in ASCs. Therefore, the simultaneous inhibition of multiple lncRNAs is expected to inhibit ASCs/BMSCs adipogenesis while promote osteogenesis.
We observe Cas13d only can surppress lncRNAs expression in rASCs. In qualitative data show both Cas13d only and Cas13d/gRNA compared with mock, there is fewer lipid drop accumulated and more calcium deposition. It seems the suppression is caused from cas13d only not Cas13d/gRNA. After using Cas13d/non-targeting gRNA as control and testing several cell lines, we decide to use mBMSCs in further experiments.
The data shows CRISPR/Cas13d system can suppress lncRNAs and adipogenesis marker genes PPARγ and CEBPα efficiently. Also it can activate osteogenesis marker genes Runx2, OSX, ALP, and OPN expression. But in Oil Red O staining, NT group still can reduce lipid drop accumulation. Show this system may still some off-target effect. 
目錄
致謝 I
摘要 II
Abstract III
第一章 文獻回顧 1
1-1骨組織工程 1
1-1-1骨骼的簡介 1
1-1-2骨組織修復重建機制 2
1-1-3骨骼組織常見傷害 4
1-1-4骨組織工程背景與發展 4
1-2幹細胞在組織工程上的應用 6
1-3 long non-coding RNAs在骨組織工程上的應用 7
1-4基因治療 9
1-5桿狀病毒表現系統 10
1-6 重組酶系統 12
1-7 桿狀病毒在骨組織工程的應用 13
1-8 CRISPR/Cas13系統 14
1-9 研究動機 16
第二章 材料與方法 26
2-1重組桿狀病毒之建構與製備 26
2-1-1昆蟲細胞培養 26
2-1-2限制酶(DNA restriction enzyme)反應及接合酶(DNA ligase)反應 26
2-1-3建構重組表現載體(donor plasmid) 26
2-1-4 DNA引子黏合反應(primer annealing) 27
2-1-5 重組表現載體之轉置反應(transposition) (Bac-to-Bac系統) 29
2-1-6重組bacmid之分離 29
2-1-7 重組bacmid之轉染反應(transfection) (製備P0病毒) 30
2-1-8 基因重組桿狀病毒放大培養 30
2-1-9超高速離心濃縮桿狀病毒 31
2-1-10桿狀病毒效價測定 31
2-2大鼠脂肪間葉幹細胞之分離與培養 32
2-3小鼠骨髓間葉幹細胞之分離與培養 32
2-4桿狀病毒轉導幹細胞之策略 33
2-5即時偵測同步定量聚合酶連鎖反應分析(qRT-PCR) 34
2-6 油紅O (Oil Red O)染色分析 36
2-7茜紅素(Alizarin Red)染色分析 37
2-8小鼠頭蓋骨植入程序 37
2-10 電腦斷層掃描分析 37
第三章 結果與討論 39
3-1 CRISPR/Cas13d系統於大鼠ASCs之測試 39
3-1-1 CRISPR/Cas13d系統之建構 39
3-1-2 Cas13d系統抑制lncRNAs效率分析 39
3-1-3以含有CRISPR array之CRISPR/Cas13d系統抑制lncRNAs基因表現分析 40
3-2 測試CRISPR/Cas13d在不同物種細胞株中的影響 41
3-3 CRISPR/Cas13d系統於小鼠BMSCs之測試 42
3-3-1 CRISPR/Cas13d系統於小鼠BMSCs之建構與評估此系統調控lncRNA表現 42
3-3-2 於mBMSC中評估CRISPR/Cas13d系統調控脂肪分化之能力 43
3-3-3 於mBMSC中評估CRISPR/Cas13d系統調控硬骨分化之能力 44
3-3-4 以Gelatin支架搭配BMSCs修復小鼠頭蓋骨缺陷之分析 44
第四章 討論 56
第五章 未來工作 58
5-1 組織切片染色分析骨組織新生與新生骨組織重塑 58
第五章 參考文獻 59

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